PCR was carried out on the DNA, using primers 4-rev and 5-rev or 14 and 15 (annealing at 58°C, 35 cycles). PCR products were visualized by gel electrophoresis

and sequences were determined through direct sequencing on the purified PCR amplicons or through cloning into pCR2.1/TOPO (Invitrogen) and subsequent sequencing with the plasmid-located primers T7 and M13 reverse. Antibiotic resistance The MIC for tetracycline was determined using E tests (BioMérieux, Boxtel, the Netherlands) on blood plates under anaerobic conditions at 37°C. Breakpoint for tetracycline was 8 μg/ml. Spectinomycin resistance was determined by an agar dilution method of C. difficile colonies on BHI agar plates, supplemented with increasing amounts of spectinomycin. Streptomycin resistance was tested by disk diffusion method, using Sensi-Neotabs (Rosco, Denmark) (Streptomycin 500 ug disks) on blood Quisinostat mw plates under anaerobic conditions at 37°C. Oligonucleotides Oligonucleotides used in this GS-1101 manufacturer study are shown in Table 3. PCR PCRs were carried out using Gotaq polymerase (Promega, Leiden, the Netherlands). Reactions contained 0.4 mM dNTPs,

0.4 uM oligonucleotides. Annealing temperature of the PCR was set at 50°C and PCRs were standardized at 30 cycles. Statistical analyses Patients samples with the full 100 kb insert were compared to patients samples with a part of the insert or no insert. The Chi-square test and t-test were used to calculate the p-value. Analyses were performed using the SPSS for Windows software package, version 17.0. MLVA Sixty eight strains were subjected to MLVA, of which 39 were previously characterized [16]. MLVA and construction of the

minimal spanning tree based on the MLVA Akt inhibitor results were carried out as described previously [16]. Acknowledgements This study was supported by HYPERDIFF-The Physiological Basis of Hypervirulence in Levetiracetam Clostridium difficile: a Prerequisite for Effective Infection Control (Health-F3-2008-223585), and by ZonMW (NWO; the Netherlands Organization for Scientific Research) grant “Reduction of community health risks of animal-associated Clostridium difficile” (project number 50-50800-98-075). APR is supported by the Medical Research Council (grant no. G0601176). Electronic supplementary material Additional file 1: Circular representation of the genome of C. difficile strain M120.The two concentric circles represent the genome (outer circle) and the G + C content (inner circle; window size 10,000; Step size 200). Green represents values higher than average (29%), purple below average. In between the two circles, the presence of the two transposable elements is indicated in red (Tn6164) and blue (Tn6190). Figure was created using DNA plotter [46]. (JPEG 39 KB) References 1. Pepin J, Valiquette L, Cossette B: Mortality attributable to nosocomial Clostridium difficile-associated disease during an epidemic caused by a hypervirulent strain in Quebec. CMAJ 2005, 173:1037–1042.